The present invention relates to the field for separating, identifying and/or analyzing particles, molecules or macromolecules, and more particularly nucleic acids, in a channel, for example in a microfluidic system or more particularly in the context of capillary electrophoresis.
Gel electrophoresis has numerous applications for separating charged particles, molecules and macromolecules, and in particular biological macromolecules such as nucleic acids (DNA, RNA, oligonucleotides), proteins, polypeptides, glycopeptides and polysaccharides. A particularly important application is sequencing, that is to say the reading of the genetic code of DNA. It is most often carried out in flat and macroscopic gels (having a thickness of the order of 1 to several mm), composed of agarose or polyacrylamide. More recently, other gels derived from acrylamide and a whole series of acrylate or methacrylate gels have been proposed for improving this or that property of the gel. In particular, in patent U.S. Pat. no. 5,164,057, there has been proposed the use of polymers having a lower critical solution temperature (abbreviated hereinafter LCST in the present application) for controlling the hydrophilic/hydrophobic character in a permanently and irreversibly crosslinked gel, used in electrophoresis.
In spite of these improvements, planar gel electrophoresis has many disadvantages.
It requires a relatively substantial amount of manual work for the preparation of the gel, which can only be used once, and for subsequent visualization of the migration distances. Reproducibility of the experiments from one gel to another is difficult to obtain because the properties of the gel depend on the exact preparation conditions. Given the production of heat linked to the current, fairly low low voltages have to be applied, leading to long separation times. Finally, planar electrophoresis is difficult to automate and to quantify. For these different reasons, macroscopic gel electrophoresis tends to be overtaken by techniques in which the separation or more generally the analysis of the constituents of a sample is carried out in channels with a high surface/volume ratio, such as cylindrical capillaries, or channels with a planar submillimetric section which are prepared in a material (which are called xe2x80x9cchipsxe2x80x9d, microchannels or microfluidic systems). Examples of such separations are given for example in xe2x80x9cCapillary electrophoresis in analytical biotechnologyxe2x80x9d, Righetti ed., CRC press, 1996, or in Cheng, J. et al., (1996), Molecular Diagnosis, 1, 183-200.
In the text which follows, all the methods involving an electrophoretic separation in one channel or several channels of which at least one of the dimensions is of a submillimetric dimension will be designated by the name xe2x80x9ccapillary electrophoresisxe2x80x9d (CE). xe2x80x9cMicrofluidic systemxe2x80x9d will designate more generally any system in which the analysis of species is carried out by virtue of the transport of the said species and/or of fluids within a channel or a set of channels of which at least one of the dimensions is submillimetric.
CE and microfluidic systems allow more rapid and higher resolution separations than gels, do not require an anticonvective medium, and their properties have been widely used to carry out separations of ions in a liquid medium.
However, a large proportion of analytes which it is desired to separate by electrophoresis, and in particular those encountered in biology, have, in a homogeneous viscous medium, a mobility which is independent of their size and can only be satisfactorily separated in a medium with obstacles. The gels used in traditional electrophoresis in particular consist of portions of macromolecular chains or of fibres which are connected to each other and cannot be crossed by the analytes. The latter have to create passages along the pores present between the macromolecular segments of the gel, which gives rise to the desired separation. In the first applications of CE to DNA and to proteins, use was often made of permanent gels which are crosslinked inside the capillary. This has many disadvantages: the preparation of a truly homogeneous gel with no bubbles inside a capillary is delicate. The gel becomes degraded relatively rapidly by hydrolysis and is xe2x80x9ccontaminatedxe2x80x9d by impurities present in the samples, which eventually damage the performance features of the separation, or even block the capillary after a limited number of separations. Given the production cost and/or the difficulty of manufacturing a capillary filled with gel, this defect makes the cost of this approach prohibitive.
Currently, the great majority of separations of biological macromolecules carried out in CE make use of solutions of entangled linear water-soluble polymers having the advantage of being replaceable as often as necessary. At a fairly high concentration, that is significantly above the entanglement threshold, the various polymers in the solution become entangled and constitute a continuous transient network of topological obstacles which cannot be crossed by the analytes, thus conferring electrophoretic separation properties on the solution.
These water-soluble polymer solutions are satisfactory in some applications, but also have numerous limitations.
The first of these limitations is electroosmosis, a simultaneous movement of the separation medium due to the presence of charges on the walls of the capillary or of the channel. As this movement is often variable over time and nonuniform, it hampers the reproducibility of the measurements and the resolution. Numerous methods have been proposed for combating it, such as treating the surface of the capillaries by adsorption of essentially neutral species on the walls of the separating channel prior to the actual separation (Wiktorowicz et al., Electrophoresis, 11, 769, 1990, Tsuji et al., J. Chromatogr. 594, 317 (1992), or by treating the capillary with an acidic solution (Fung et al., Anal. Chem. 67, 1913, (1995)). These methods have the advantage of being inexpensive and of being capable of being repeated several times in order to regenerate a capillary, but often only partially reduce the electroosmosis. Methods for the irreversible grafting of an essentially neutral polymeric layer on the walls have also been proposed, as for example described in U.S. Pat. No. 4,680,201. Ready-to-use treated capillaries are thus commercially available. These irreversibly treated capillaries lead to a good reduction of electroosmosis for a certain number of separations, but their shelf life is limited and their cost is high.
Another major disadvantage of electrokinetic separations in polymer solutions is that the resolution and the separable range of sizes are better with solutions which are relatively concentrated and of high molecular masses (Mitnik et al., J. Chrom. A, 710, 309 (1995); Goetzinger et al., Electrophoresis, 19, 242, 1998)). This is attributed to deformations of the separating matrix which limits the resolution for large size analytes and which are greater the lower the molecular mass of the matrix and the lower its concentration. On the other hand, the viscosity of a polymer solution increases very rapidly when the molecular mass and the concentration are increased. The application of entangled water-soluble polymer solutions is therefore limited by the very great difficulty, and as a last resort the impossibility, of introducing into a small size capillary (typically less than 100 micrometers) a solution of very high viscosity. Finally, it should be noted that the separation range accessible to capillary electrophoresis may be extended to the largest sizes by the use of pulsed fields. Phenomena of DNA aggregation are encountered in this case which limit the scope of the improvement, and which are also greater, the lower the viscosity of the medium.
To solve the dilemma posed by the search for a low viscosity for injecting the separation medium into the channel, and for resistant topological obstacles for the separation, which in fact lead to a high viscosity, some authors have proposed using a polymer medium whose viscosity decreases considerably during a rise in temperature. This type of medium has the advantage of allowing the injection of the said medium into the capillary at high temperature in a low viscosity state, and separation at a lower temperature in a higher viscosity state exhibiting good separation performance features, as is commonly carried out in gel electrophoresis, in particular with agarose.
In applications WO 94/10561 and WO 95/30782, media are proposed in particular which allow easier injection by raising the temperature. Essentially described in these patent applications are microgels capable of decreasing in volume at high temperature (thus leading to a dilute solution of discontinuous particles of low viscosity) and of swelling at low temperature to the extent of fully occupying the separating channel (thus conferring a gelled character and good separation properties on the medium).
However, these separation media have a viscosity which decreases more or less rapidly with temperature: it is therefore necessary to introduce them into the capillary at a temperature greater than the temperature at which the separation occurs, which may have various disadvantages. On the one hand, in capillary electrophoresis apparatus, it is very difficult to thermostat the entire capillary, and it is therefore difficult to use in an automated fashion a polymer which would only be injectable at a temperature which is markedly greater than room temperature. It would be possible to envisage a solution which is not very viscous at room temperature, and which has a high viscosity and good separation properties at a lower temperature, but that involves carrying out the separations at low temperatures, which is not possible for all analytes. In particular, it is known that for DNA sequencing, an optimum resolution of the xe2x80x9ccompressionsxe2x80x9d is obtained at a relatively high temperature (of the order of 50-60xc2x0 C.), which is incompatible with the preceding principle.
Application WO 98/10274 proposes, for its part, a molecular separation medium comprising at least one type of block copolymer which is in solution at a first temperature and in a gel-type state at a second temperature. This medium comprises, in addition, a buffer whose role is to dissolve the block copolymer at a first temperature, and to cause it to transit towards the gel state at the said second temperature without interrupting the separation process, and without preventing a return to a soluble state during the return to the said first temperature. More specifically, the polymers described are triblock polymers of low molecular massses (typically less than 20,000), of the polyoxyethylene-polyoxypropylene-polyoxyethylene (POE-POP-POE) family and more specifically still (POE99-POP69-POE99, where the subscripts represent the numbers of monomers of each block) (trade name xe2x80x9cPluronic F127xe2x80x9d). At low temperature, the two POE segments at the ends of the triblock systems are water-soluble, and given the low molecular mass of the copolymer, the solutions are relatively only slightly viscous up to a high concentration. By raising the temperature to around 15-25xc2x0 C., the central POP central segment becomes more hydrophobic, and these polymers combine to constitute inside the medium an organized three-dimensional network having a predetermined structure, which confers the appearance and the consistency of a gel on the medium. Unfortunately, this mechanism exhibits several disadvantages for electrophoresis. On the one hand, it only gives rise to a gel state endowed with good electrophoretic separation properties at high polymer concentrations, greater than 15 g/100 ml or even 20 g/100 ml, which leads to high friction and to long migration times. Moreover, the dependence of the properties as a function of the rate of change in temperature makes the reproducibility of the results unpredictable. Finally, the products proposed in WO 98/10274 exhibit a low viscosity either below room temperature or above room temperature, and exhibit a gel state which is advantageous for electrophoretic separation in the region of room temperature (25xc2x0 C.), which is not advantageous either for conveniently filling the capillaries, or for applications such as DNA sequencing.
Triblock copolymers having the same structure, with different molecular masses, give rise to comparable physical properties and to performance features in terms of separation which are comparable to or less than F127. Also described in WO 98/10274 is a POB12-POE260-POB12-type polymer, where POB means polyoxybutylene. Unlike the preceding polymers, these polymers give rise to a state which is not very viscous at a temperature greater than room temperature, and to gelling by lowering of the temperature to the region of room temperature.
It should be noted that other polymers exist which may exhibit a thermoviscosity-promoting or thermothickening character in water. Thermoviscosity-promoting polymers are described in Patent Application EP 583 814 which contain, on the one hand, hydrophilic portions of the prepolymer or macromonomer chain type which do not exhibit an LCST in a useful temperature range, and, on the other hand, hydrophilic portions of the prepolymer or macromonomer chain type which exhibit an LCST in the said useful temperature range. However, polymers of this type cannot be used with good performance features as separation medium for electrophoresis for several reasons. On the one hand, they only exert their thermoviscosity-promoting effect in the presence of a relatively large quantity of salt in the solution, between of the order of 0.4 M and several M. This property is highly crippling for electrophoresis, because the use of highly saline solutions leads to heating of the solution and prevents the use of strong electric fields. The separations in highly saline media are therefore slow and of low resolution. Moreover, they exhibit an electrically charged skeleton: if it is desired to use them as electrophoretic separation medium, the polymers constituting the matrix risk being set in motion, or giving rise to electroendoosmosis, disrupting the separation which it is desired to obtain through interaction between the analytes and fixed obstacles. Thirdly, these media are intended to give rise to thermoviscosification or to maintain a roughly constant viscosity in a unique and relatively broad temperature range covering the LCST of the said hydrophilic portions with LCST. However, it would be particularly advantageous, for electrophoresis, to have two clearly distinct useful temperature ranges, one for injecting the separation medium into the capillary, and the other for the actual electrophoretic separation, the said separation being carried out while the medium is maintained at a roughly constant temperature.
In fact, most synthetic media proposed up until now as being capable of giving rise to a thermoviscosifying effect have a charged skeleton (L""Alloret et al., Colloid. Polym. Sci., 273, 1163-1173 (1995), Hourdet, Polymer preprints, 34, 972-973 (1993), Hourdet et al., Polymer, 38, 2535-2547 (1997)). Indeed, a customary way of conferring thermoviscosifying properties on a polymer consists in constructing a molecule having chain portions which are hydrophilic at any temperature and which help to maintain the molecule in solution, and chain portions with LCST which lead, through a rise in temperature, to an attractive interaction between chains, which are responsible for the viscosification. To obtain good viscosification, it would be desirable to multiply the proportion of chains with LCST and their force of interaction, but such an increase also tends to induce a macroscopic phase separation which, by contrast, reduces the viscosity. The presence of electrical charges on the skeleton helps to prevent the macroscopic phase separation through an effect of entropy of the counter-ions or of electrostatic repulsion, and therefore makes it possible to cause entry into the polymer composition of a quantity of portions with LCST which is sufficient to give viscosification, while preventing macroscopic phase separation. It can therefore be seen that it is particularly difficult to constitute thermoviscosifying media based on polymers with a neutral hydrophilic skeleton. A few examples of such media have been described in Vos et al., Polymer, 35, 2644 (1994), but they only give rise to thermoviscosification in the presence of a high salt level and at temperatures of more than 80xc2x0 C., which also makes them unusable for most of the applications which may be envisaged as a separating matrix, and in particular for electrophoresis.
Consequently, although many types of media, heat sensitive or otherwise, have been proposed as matrices for the electrokinetic separation of species in a channel, and numerous types of heat-sensitive media have also been proposed for other applications, there are currently no media exhibiting optimum properties for the said separations.
The object of the present invention is precisely to provide a novel type of separation media for which it is found to be possible to optimize the properties as a function of the size of the analytes which it is desired to separate through the selection of a specific copolymer.
For the purposes of the invention, the expression separation is intended to cover any method aimed at separating, identifying or analyzing all or some of the species contained in the mixture, the said species being commonly called xe2x80x9canalytesxe2x80x9d.
This separation may thus be carried out in a channel in a microfluidic system or in the context of electrophoresis.
The invention is particularly advantageous in the case of electrokinetic separations.
The expression electrokinetic separation is intended to cover any method aimed at separating all or some of the species contained in a mixture, the said species being commonly called xe2x80x9canalytesxe2x80x9d, by causing them to migrate in a medium under the action of an electric field, whether the field exerts its moving action on the analytes in a direct or indirect manner, for example via a movement of the medium itself, as in electrochromatography, or a movement of ancillary species such as micelles, in the case of micellar electrochromatography, or by any combination of direct or indirect actions. Any method of separation in which the said action of the electric field is combined with another moving action of a nonelectrical origin will also be considered as an electrokinetic separation method according to the invention.
More particularly, the subject of the present invention is a heat-sensitive medium for the separation of species in a separating channel, the said medium comprising an electrolyte in which at least a set of block copolymers is dissolved, characterized in that the said block copolymers:
are provided in the said electrolyte at a sufficient concentration to confer on the said medium the ability to reversibly transit from a viscosity state V1, obtained at a temperature. T1, to a viscosity state V2 which is at least 100% higher than V1, obtained at a temperature T2 which is at least 20xc2x0 C. higher than T1 and
comprising in their structure at least:
two noncontiguous polymeric segments exhibiting an LCST in the said electrolyte and possessing an average number of atoms along their skeleton which is greater than 50, and
a polymeric segment which is soluble in the electrolyte at the temperatures T1 and T2.
For the purposes of the invention, unless otherwise explicitly stated, all the average values on sets of chains or on sets of polymeric segments, such as the average molecular mass, or the average number of atoms along the skeleton, or alternatively the average number of grafts in the case of a comb polymer, are understood to mean average values by mass within the usual meaning of polymer physics.
The separation medium claimed therefore possesses the capacity to reversibly transit between a fluid state with a fairly low viscosity so as to allow its introduction into the said channel, obtained at a temperature T1, and a state with a markedly higher viscosity, and in any case at least twice as high, obtained at a temperature T2 which is at least 20xc2x0 C. higher than the temperature T1. At the temperature T2, the said separation medium is endowed with significant separating properties for species in a predefined range of chemical composition and size.
The expression xe2x80x9ctemperature T1xe2x80x9d is understood to mean, in the context of the invention, either a precise temperature, or a relatively narrow range of temperatures, of a level which is typically of the order of 10xc2x0 C., useful for carrying out a particular operation relating to the separation process, and in particular for introducing the separating matrix according to the invention into the separating channel. According to a preferred variant of the invention, the temperature T1is between about 15 and 30xc2x0 C. Likewise, the expression xe2x80x9ctemperature T2xe2x80x9d is understood to mean, in the context of the invention, either a precise temperature or a relatively narrow range of temperatures, of a level which is typically of the order of 10xc2x0 C., useful for carrying out another particular operation relating to the separation process, and in particular for the step for separating analytes in the channel. According to a preferred variant of the invention, useful for the sequencing of DNA, this temperature or temperature range T2 is between about 40 and 80xc2x0 C.
According to a preferred variant of the invention, the LCST of a significant fraction of the said segments exhibiting an LCST is between the temperatures T1 and T2 and more preferably between about 20 and 50xc2x0 C.
For the purposes of the invention, the expression electrolyte is understood to mean a condensed medium capable of conducting ions. In the most common case, this medium is a buffered aqueous medium, such as buffers based on phosphate, tris(hydroxymethyl)amino-methane (TRIS), borate, N-tris(hydroxymethyl)methyl-3-aminopropanesulphonic acid (TAPS), histidine, lysine, and the like. Numerous examples of buffers which can be used in electrophoresis are known to persons skilled in the art, and a number of them are described for example in Sambrook et al., xe2x80x9cMolecular Clonning: a laboratory manualxe2x80x9d, Cold Spring Harbor Lab, New York, 1989. However, any type of electrolyte may be used in the context of the invention, in particular aqueous-organic solvents such as, by way of example, water-acetonitrile, water-formamide or water-urea mixtures, polar organic solvents such as, again by way of example, N-methylformamide. Particularly useful in the context of the invention are the electrolytes termed xe2x80x9csequencing buffersxe2x80x9d, consisting of an aqueous buffer at alkaline pH supplemented with a substantial proportion of urea and/or of formamide.
For the purposes of the invention, the term xe2x80x9cspeciesxe2x80x9d is understood to mean analytes in general. These analytes may be particles, organelles or cells, molecular or macromolecular species, and in particular biological macromolecules such as nucleic acids (DNA, RNA, oligonucleotides), nucleic acid analogues obtained by chemical modification or synthesis, proteins, polypeptides, glycopeptides and polysaccharides whose complete or partial separation is desired during their electrokinetic migration within the said separation medium.
For the purposes of the invention, the expression xe2x80x9cpolymeric segmentxe2x80x9d or xe2x80x9csegmentxe2x80x9d is understood to mean a set of monomers covalently linked to each other and having specific physicochemical properties, in particular as regards solvation. One example of a polymeric segment for the purposes of the invention is given by a chain of monomers which are all identical (homopolymeric segment), or a copolymer exhibiting no significant correlation in composition over lengths of more than a few monomers (segment of the random copolymer type).
For the purposes of the invention, the expression block copolymer is understood to mean a copolymer consisting of polymeric segments having significantly different compositions, covalently linked to each other. The block copolymer is defined by the fact that each of the segments comprises a sufficient number of monomers so as to exhibit in the electrolyte physicochemical and, in particular, salvation properties which are comparable to those of a homopolymer having the same composition and the same size. It is distinguishable from the random polymer in which the various types of monomer follow each other in an essentially random manner, and locally confer overall properties on the chain which are different from those of the homopolymers of each of the species in question. The size of the homopolymeric segments necessary for obtaining this block character may vary according to the types of monomer and the electrolyte, but it is typically a few tens of atoms along the skeleton of the said segment. It should be noted that it is possible to constitute a block copolymer within the meaning of the invention, in which some or all of the segments themselves consist of a random-type copolymer in so far as it is possible to distinguish within the said block copolymer zones or segments having a size and a difference in chemical composition which are sufficient to give rise, from one segment to another, to a significant variation in the physicochemical, and in particular salvation, properties. Finally, the expression xe2x80x9cnoncontiguous polymeric segmentsxe2x80x9d within a block polymer is understood to mean two segments linked to each other by a polymeric segment of a different nature.
The block copolymers suitable for the invention possess the feature of combining at least two types of segment in their structures.
The first type of segment is soluble in the electrolyte used for the separation at the two temperatures T1 and T2 for using the claimed medium and preferably do not exhibit an LCST in the said electrolyte. In the present context, the term soluble is understood to refer to solubility in the electrolyte at the temperatures T1 and T2.
On the other hand, the second type of segments is endowed with an LCST in the electrolyte used for the separation. More precisely, this type of segment is essentially soluble in the said electrolyte in a range of low temperatures, and is essentially insoluble in the said electrolyte in a range of high temperatures. The limit between these two temperature ranges is called xe2x80x9cminimum demixing temperaturexe2x80x9d or more commonly xe2x80x9cLCSTxe2x80x9d.
By virtue of the presence of these segments with LCST at the level of their structure, the copolymers used according to the invention possess the property of constituting, at low temperature, a macroscopically homogeneous entangled solution in which the interactions between various polymer molecules are essentially repulsive, and of giving rise, following an increase in temperature, to attractive interactions between some of their parts which strengthen the disentangling interactions between chains.
According to a preferred embodiment of the invention, the average number of segments with LCST per copolymer is greater than 2 and preferably greater than 5, and more preferably still of between 8 and 40. By virtue of this large number of segments, capable of interacting in an attractive manner, a given copolymer may interact with numerous other copolymers, which confers high resistance on the medium during the passage of analytes.
The length and the number of segments with LCST present in the copolymers used in the media according to the invention, as well as their chemical nature, may consequently vary significantly in the context of the invention, and may thus greatly cause the viscoelastic properties of the said media to vary considerably according to the desired application, as will be shown more precisely on disclosing the examples of implementation.
The media according to the invention are therefore suitable for producing thermothickening properties. For the purposes of the invention, xe2x80x9cthermothickeningxe2x80x9d refers to a medium having either a thermoviscosifying character, or a thermogelling character.
In accordance with common usage, the term thermoviscosifying is understood to mean a medium remaining in the temperature range(s) for use which is capable of flowing in a macroscopic vessel, in a time compatible with easy handling, that is less than about 30 seconds. By contrast, the expression xe2x80x9cgel-type statexe2x80x9d is understood to mean a medium incapable of significant flow under the same conditions.
In an equivalent manner, the term thermoviscosifying refers to a medium which does not exhibit in the temperature range(s) for use hysteresis of their properties, or a significant dependence of its properties as a function of the rate of change of temperature, for rates of change of temperature customarily and conveniently used in capillary electrophoresis, that is of the order of 1 degree per minute to about ten degrees per minute. On the other hand, the expression xe2x80x9cthermogelling mediumxe2x80x9d, or in an equivalent manner xe2x80x9cmedium giving rise to a gel-type statexe2x80x9d will refer to a medium exhibiting, under these conditions, hysteresis of its properties or else possessing a viscosity greater than 20000 cp.
According to a preferred variant which is particularly suited to the case of automated methods in which it is desired to minimize the time between two consecutive separations, the medium according to the invention will be of the thermoviscosifying type.
According to another preferred variant, when a long waiting time between separations is acceptable and when a maximum resistance of the matrix to the passage of the analytes is desired, a medium according to the invention which gives rise at the temperature T2 to a gel-type state will on the contrary be preferred.
According to a preferred embodiment of the invention, all or some or a significant fraction of the segments with LCST possess along the skeleton an average number of atoms greater than 75, or have a molecular mass greater than 2500, and preferably greater than 4500.
For the purposes of the invention, the expression xe2x80x9ca significant fractionxe2x80x9d, or in an abbreviated form xe2x80x9call or somexe2x80x9d, is understood to mean a proportion sufficient to give rise, through an increase in temperature, to an increase in viscosity of at least 100% or in an equivalent manner to the multiplication of the viscosity by a factor of 2.
Optimized copolymers for carrying out the invention are in particular those in which the set of segments with LCST represents between 2 and 25% by mass, preferably between 5 and 15% by mass and more preferably still between 8 and 15% by mass of the average total molar mass of the said copolymers, or between 3 and 20% and preferably between 5 and 10% of the total compositon of the copolymers as number of moles of monomers.
The claimed separation medium may advantageously comprise a set of block copolymers comprising a skeleton consisting of a segment or a multiplicity of segments, of an identical or different chemical nature, and exhibiting the common character of being essentially soluble or else solvated in the electrolyte at the temperatures T1 and T2, to which there are covalently linked a multiplicity of segments of an identical or different chemical nature and exhibiting the common characteristic of being essentially soluble or else solvated in the electrolyte at the temperature T1, and essentially insoluble or poorly solvated in the electrolyte at the temperature T2.
All sorts of structures of copolymers of this type may be used for carrying out the invention provided that they have a multiplicity of segments with LCST which are not directly connected, and give rise to reversible thermothickening under the separation conditions.
By way of illustration of various structures which may be adopted by the copolymer according to the invention, there may be mentioned most particularly those where all or part of the said copolymers exist:
in the form of a linear block polymer,
in the form of a comb copolymer whose skeleton consists of one or more segments which are soluble in the electrolyte at the temperatures T1 and T2 or else
in a branched form.
In the linear block polymer structure, the segments with LCST are present in a number greater than 2 and are separated from each other along an essentially linear skeleton by polymer segments which do not have an LCST. The length of the latter segments may be, in the context of the invention, either relatively uniform or on the contrary variable, the latter variant being preferred.
In fact, it is particularly advantageous to choose a block polymer of the comb polymer type, with a skeleton which is essentially soluble in the electrolyte at the temperatures T1 and T2, carrying a multiplicity of side members which are essentially soluble in the electrolyte at the temperature T1 and insoluble in the electrolyte at the temperature T2. According to a preferred embodiment, the said side members with LCST are arranged along the skeleton in a random or irregular manner.
This embodiment is in general preferable to the opposite structure, which would comprise polymers consisting of a main chain consisting of segments with LCST carrying hydrophilic grafts, in so far as a polymer of this second type contracts above the LCST and thus cannot give rise to a continuous network of topological obstacles necessary for carrying out the invention.
It should be noted that the invention cannot be carried out with block polymers consisting of segments soluble in the electrolyte and of hydrophobic segments without LCST, since such polymers give rise to thermofluidification.
Particularly advantageous in the context of the present invention are separation media in which at least one of the following conditions is satisfied:
all or some of the copolymers possess an average number of atoms, along a soluble section of segment between two consecutive points for binding of said soluble segment with segments with LCST, greater than 210;
all or some of the copolymers possess a molecular mass greater than 30000 or a number of atoms along the main skeleton greater than 2000, and
all or some of the copolymers possess a molecular mass between 50000 and 3000000 or a number of atoms along the main skeleton between 2500 and 100000, and/or
the average number of segments with LCST per chain is greater than or equal to 4, and is preferably between 5 and 100.
It is particularly advantageous, for carrying out the invention, to use copolymers in which the segment(s) soluble at the temperatures T1 and T2 consist of at least one polymer chosen from polyethers, polyesters such as polyglycolic acid, soluble random copolymers and homopolymers of the polyoxyalkylene type such as polyoxypropylene, polyoxybutylene, polyoxyethylene, polysaccharides, polyvinyl alcohol, polyvinylpyrrol-idone, polyurethanes, polyamides, polysulphonamides, polysulphoxides, polystyrenesulphonate, substituted or unsubstituted polyacrylamide or polymethacrylamide derivatives soluble in the said electrolyte.
By way of illustration of the polyacrylamides and polymethacrylamides, there may be mentioned most particularly polyacrylamide, polyacrylic acid, poly(N,N-dimethylacrylamide) and polyacryloylamidopropanol.
Of course, other polymers soluble in the electrolyte may be used according to the invention as soluble segments, according to the particular application and the ease of introducing them into a block polymer with the desired structure.
It is more generally advantageous for the soluble segments to have a high solvation in the electrolyte at the two temperatures T1 and T2.
Numerous types of polymer may be chosen to constitute the noncontiguous segments with LCST inside a block copolymer which can be used according to the invention, according to the electrolyte envisaged, the preferred temperatures of T1 and T2 for the implementation and the analytes to be separated. Numerous polymers with LCST are known to the person skilled in the art, in particular in aqueous medium. Reference may thus be made to the book xe2x80x9cPolymer Handbookxe2x80x9d Brandrupt and Immergut, Johy Wiley, New York.
According to a preferred variant of the invention, all or some of the polymeric segments with LCST are derived from one or more polymers chosen from:
polyvinyl alkyl ethers such as polyvinyl methyl ether,
hydroxyalkyl celluloses such as hydroxyethyl cellulose and methyl cellulose,
homopolymers of ether oxides like polyoxyalkylenes such as polyoxypropylene and polyoxybutylene,
random and block copolymers of ether oxides such as copolymers of the polyoxyalkylene type having an LCST like polyoxyethylene/polyoxypropylene and polyoxyethylene/polyoxybutylene,
alkylene homo- and copolymers like butylene-propylene, ethylene-propylene and ethylene-butylene, and
polyacrylic derivatives derived from the homopolymerization or copolymerization of monomers chosen from acrylic and methacrylic acids, alkyl acrylates and methacrylates such as hydroxypropyl and hydroxyethyl acrylates, N-alkyl-acrylamides or -methacrylamides such as N-ethylacrylamide, N-isopropylacrylamide, Nxe2x80x2,N-dialkyl-acrylamides or -methacrylamides, aryl-acrylamides or -methacrylamides and alkylaryl-acrylamides or -methacrylamides.
More preferably, the copolymer used according to the invention comprises at least two noncontiguous segments with an LCST property derived from the homo- or copolymerization of monomers chosen from acrylic and methacrylic acids, N-alkyl-acrylamides or -methacrylamides such as N-ethylacrylamide, N-isopropylacrylamide, aryl-acrylamides or -methacrylamides and alkylaryl-acrylamides or -methacrylamides.
Thus, it is possible to use, by way of example and without limitation, as segments with LCST, N-isopropyl-acrylamide (NIPAM), N-isopropylmethacrylamide, N,Nxe2x80x2-diethylacrylamide, or random copolymers of these monomers with each other or with others.
The following copolymers are most particularly suitable for the invention:
copolymers of the comb copolymer type whose skeleton is of the type including acrylamide, acrylic acid, acryloylaminoethanol or dimethacrylamide and on which there are grafted side segments of the poly(N-alkyl or N,N-dialkyl)acrylamide type, preferably of the poly(N-isopropylacrylamide) type, or side segments of the random or block, polyoxyethylene/oxypropylene copolymer or polyoxypropylene type, or more generally side segments of the polyether type which are notably more hydrophobic than polyoxyethylene
copolymers of the block copolymer type exhibiting along their skeleton an alternation of segments of the polyoxyethylene type and of segments of the polyoxypropylene type, or an alternation of segments of the polyoxyethylene type and of segments of the polyoxybutylene type or more generally an alternation of segments of polyethylene and of segments of the polyether type which are notably more hydrophobic than polyoxyethylene.
By way of examples, it is possible to use, as copolymers, in the claimed separation medium, copolymers chosen from polyacrylamide/poly(N-isopropylacrylamide) (PAM-NIPAM); polyvinylalcohol/poly(N-isopropylacrylamide) (PVA-NIPAM), polyoxyethylene/polyoxypropylene, polyacrylamide/oxyethylene-oxypropylene copolymer, polyacrylamide/polyoxypropylene, polyacrylic acid/polyoxypropylene, polyacrylic acid/oxyethylene-oxypropylene copolymer, polyacrylic acid/poly(N-isopropylacrylamide) and polydimethylacrylamide/poly(N-isopropylacrylamide) (PDMAM-NIPAM).
It is possible to advantageously use a medium comprising a comb copolymer carrying along the polyacrylamide skeleton segments with LCST essentially consisting of poly-NIPAM, and comprising along their skeleton a number of carbon atoms between 35 and 600, and the total mass fraction of which does not exceed 25%.
It should also be noted that in the majority of applications, and in particular for separating charged analytes, it is preferable to use a polymer according to the invention which is essentially neutral. It may however be useful in some applications, and in particular for separating weakly charged or uncharged analytes, or analytes which tend to combine with the polymer, to choose a polymer according to the invention which is deliberately charged. It will be possible to conveniently prepare polymers of this type, for example, by allowing a substantial portion of polymerized acrylic acid to be present in the segments soluble in the electrolyte at the temperatures T1 and T2. A copolymer of this type is more particularly described in Example 10 below.
As regards more particularly the copolymer concentration in the medium, it is generally less than 20 g/100 ml by weight. For DNA sequencing applications, it is preferably between about 1 and 8 g/100 ml by weight.
According to a particular embodiment of the invention, the copolymers used in the said medium are advantageously capable, from a concentration of the order of 5 g/100 ml and preferably of the order of 2 g/100 ml by weight, of causing the said medium to reversibly transit from a viscosity state V1, obtained at a temperature T1, to a viscosity state V2 which is at least 100% greater than V1 and which is obtained at a temperature T2 which is at least 20xc2x0 C. greater than T1.
Also according to a preferred variant of the invention, the viscosity V2 is greater than the viscosity V1 by a factor of at least 5.
As regards the preparation of the copolymers used according to the invention, it may be carried out by any conventional polymerization or polycondensation technique. The choice of the method of preparation is generally carried out while taking into account the structure desired for the copolymer, namely a comb, linear or branched structure, and the chemical nature of the various blocks constituting it.
By way of illustration of these variant preparations, there may be mentioned most particularly the processes according to which the said copolymers are obtained by:
ionic or free-radical polycondensation, polymerization or copolymerization of identical or different monomers, of identical or different macromonomers, or of a mixture of identical or different monomers and macromonomers, or
by grafting several polymeric segments with LCST onto a linear or branched polymeric skeleton which is essentially of the soluble type, or by polymerization of polymeric side segments with LCST from a linear or branched polymeric skeleton which is essentially of the soluble type.
Preferably, all or some of the copolymers used according to the invention are obtained by:
a) copolymerization of monomers which are essentially of the soluble type and of macromonomers which are essentially of the type with LCST comprising a reactive functional group at least at one of their ends, or
b) copolymerization of macromonomers which are essentially of the type with LCST comprising a reactive functional group at least at one of their ends, and macromonomers which are essentially of the soluble type comprising at least one reactive functional group in their structure.
For the purposes of the invention, the expression reactive functional group is understood to mean a group allowing the molecule carrying this group to be integrated into the macromolecule during the copolymerization reaction without interrupting the said copolymerization.
Using the preferred methods and rules set out above, persons skilled in the art are capable of preparing copolymers in accordance with the invention by adapting the structure, the nature and the method of preparation of the said polymers according to the separation properties desired for one application or another.
By way of nonlimiting illustration of the claimed separation media mention may be most particularly be made of the following media:
the media transiting from a viscosity V1 of between 50 and 1000 mPa.mxe2x88x921 at a temperature T1 of between 15 and 30xc2x0 C. to a viscosity V2 which is greater than V1 by a factor of between 2 and 50 at a temperature T2 of the order of 40xc2x0 C. or higher and comprising between 5 g/100 ml and 20 g/100 ml of copolymers possessing
an average molecular mass of between 30000 and 2000000 or a number of atoms along the main skeleton of between 1000 and 60000,
a fraction by mass of segments with LCST of between 2% and 20%, and
an average molecular mass of the segments with LCST of between 2000 and 20000 or an avarage number of atoms along a segment with LCST of between 35 and 350;
the media transiting from a viscosity V1 of between 100 and 10000 mPa.mxe2x88x921 at a temperature T1 of between 15 and 30xc2x0 C. to a viscosity V2 which is greater than V1 by a factor of between 2 and 100 at a temperature T2 higher than 40xc2x0 C. and comprising between 1 g/100 ml and 80 g/100 ml of copolymers possessing:
an average molecular mass of between 500000 and 5000000 or a number of atoms along the main skeleton of between 7000 and 90000,
a fraction by mass of segments with LCST of between 2.5% and 15%, and
an average molecular mass of segments with LCST of between 4000 and 30000 or an average number of atoms along a segment with LCST of between 60 and 600; and
the media transiting from a viscosity V1 of between 100 and 10000 mPa.mxe2x88x921.sxe2x88x921 at a temperature T1 of between 15 and 30xc2x0 C. to a viscosity V2 which is greater than V1 by a factor of between 2 and 100 at a temperature T2 of the order of 40xc2x0 C. or higher and comprising between 0.1 g/100 ml and 5 g/100 ml of copolymers possessing
an average molecular mass greater than 500000 or a number of atoms along the main skeleton greater than 7000,
a fraction by mass of segments with LCST of between 2% and 15%, and
an average molecular mass of the segments with LCST greater than 4000 or an average number of atoms along a segment with LCST greater than 90.
In the present description, the viscosity is that obtained at a shear rate of 10 sxe2x88x921.
It is also possible, in the context of the invention, to include in the separation medium, in addition to the copolymer(s) with a thermothickening character, other species not exhibiting these properties, such as in particular water-soluble polymers, nonthermothickening associative polymers, or else neutral or ionic surfactants, provided that these adjuvants do not give rise to demixing in the separation medium or to a loss of the reversible thermothickening character. Such adjuvants may be advantageous for modulating the properties and/or the separating power of the said medium. It is in particular known that the addition of certain surfactants may in some cases reinforce the association between polymers, and therefore the thermothickening character. It may also be advantageous to add to the medium associative or nonassociative polymers of low molecular weight in order to enhance the separation of the smallest analytes contained in a mixture without adversely affecting the overall viscosity, as is known in contexts different from that of the invention.
The subject of the present invention is also the use of a separation medium as defined above for the separation or analysis of species among molecular or macromolecular species, and in particular biological macromolecules such as nucleic acids (DNA, RNA, oligonucleotides), nucleic acid analogues obtained by chemical synthesis or modification, proteins, polypeptides, glycopeptides and polysaccharides, organic molecules, synthetic macromolecules or particles such as mineral particles, latex, cells or organelles.
It is particularly useful for the sequencing of DNA, for which it makes it possible to obtain optimum separation conditions linked to a high viscosity, to a high temperature for which the resolution of compressions is good and the separation rapid, while preserving, at room temperature, a moderate viscosity allowing easy injection of DNA.
However, the possibility offered by the invention of considerably varying the viscosity of the medium by changing temperature is also advantageous for other applications.
It is in particular advantageous every time an increase in the rigidity or the viscosity of the medium contained in a capillary or a microchannel might be desirable. For example, a high viscosity makes it possible to reduce the electrohydrodynamic effects responsible for a poor separation of large DNAs in a pulsed field and more generally to reduce the damaging hydrodynamic flows in a capillary or a microchannel.
It should also be noted that although the media according to the invention in general best exert their beneficial effect by means of a change in temperature, they are also suitable for use at constant temperature. They may thus be suitable for introduction into the separating channel and for the consecutive separation of analytes at the same temperature. This may be advantageous in particular if an apparatus is available which is capable of introducing into the separating channel a medium of high viscosity, and/or which does not make it possible to easily modify the temperature between the introduction of the said medium into the channel and the separation of the analytes.
Another advantage of the high viscosities allowed by the media according to the invention is that they reduce electroosmosis (see for example Bello et al., Electrophoresis 15, 623, 1994), without a need to use other methods for suppressing electroosmosis known to persons skilled in the art, such as for example the use of polymers having affinity for the wall of the capillary, or else the grafting of neutral hydrophilic polymers onto the surface. If the suppression of electroosmosis or the interaction of the analytes with the walls produced by the media according to the invention is not sufficient for a particular application, it is possible to combine the invention with one of these processes known to persons skilled in the art, and to thus obtain properties which are even better than those obtained with these methods used alone.
Advantageously, it is possible to modulate the separating properties of the claimed medium via the selection of a copolymer in accordance with the invention and whose thermothickening effect is more particularly optimized for the separation of species of different sizes.
By way of illustration of this type of adaptation which is accessible according to the invention, it is possible in particular:
to separate molecules having a molecular mass of less than 50000 or oligonucleotides comprising less than 100 nucleotides, or else native or denatured proteins with a medium transiting from a viscosity V1 of between 50 and 1000 mPa.mxe2x88x921.sxe2x88x921 at a temperature T1 of between 15 and 30xc2x0 C. to a viscosity V2 which is greater than V1 by a factor of between 2 and 50 at a temperature T2 of the order of 40xc2x0 C. or higher and comprising between 5 g/100 ml and 20 g/100 ml of copolymers possessing:
an average molecular mass of between 30000 and 2000000 or a number of atoms along the main skeleton of between 1000 and 60000,
a fraction by mass of segments with LCST of between 2% and 20%, and
an average molecular mass of the segments with LCST of between 2000 and 20000 or an avarage number of atoms along a segment with LCST of between 35 and 350,
to separate products of reaction of DNA sequence, DNA duplexes of less than 1000 base pairs, denatured proteins or synthetic or natural polymers having a molecular mass of between 20000 and 10000000 with a medium transiting from a viscosity V1 of between 100 and 10000 mpa.mxe2x88x921.sxe2x88x921 at a temperature T1 of between 15 and 30xc2x0 C. to a viscosity V2 which is greater than V1 by a factor of between 2 and 100 at a temperature T2 higher than 40xc2x0 C. and comprising between 1 g/100 ml and 8 g/100 ml of copolymers possessing:
an average molecular mass of between 500000 and 5000000 or a number of atoms along the main skeleton of between 7000 and 60000,
a fraction by mass of segments with LCST of between 2.5% and 15%, and
an average molecular mass of segments with LCST of between 4000 and 30000 or an average number of atoms along a segment with LCST of between 60 and 600 or
to separate DNA duplexes having a size of between 500 bases and several millions of base pairs, or particles such as latexes, whole cells, whole chromosomes or organelles with a medium transiting from a viscosity V1 of between 100 and 10000 mPa.mxe2x88x921.sxe2x88x921 at a temperature T1 of between 15 and 30xc2x0 C. to a viscosity V2 which is greater than V1 by a factor of between 2 and 100 at a temperature T2 of the order of 40xc2x0 C. or higher and comprising between 0.1 g/100 ml and 5 g/100 ml of copolymers possessing:
an average molecular mass greater than 500000 or a number of atoms along the main skeleton greater than 7000,
a fraction by mass of segments with LCST of between 2% and 15%, and
an average molecular mass of the segments with LCST greater than 4000 or an average number of atoms along a segment with LCST greater than 90.
More preferably, these media comprise a set of copolymers chosen from:
copolymers of the comb copolymer type whose skeleton is of the acrylamide, acryloylamino-ethanol or dimethylacrylamide type and on which side chains of the poly(N-isopropylacrylamide) type are grafted, and
copolymers of the block copolymer type and which exhibit along their skeleton an alternation of blocks of the polyoxyethylene type and blocks of the polyoxypropylene type, or an alternation of blocks of the polyoxyethylene type and of blocks of the polyoxybutylene type, or more generally an alternation of blocks of the soluble polyoxyalkylene type and of polyoxyalkylene blocks with LCST.
By way of illustration of the method of using the claimed separation medium there may be proposed in particular that comprising:
selecting a separation medium according to the invention, according to the characteristics of the species to be separated;
introducing this medium into a separating channel of an electrophoresis apparatus in a sufficient quantity to constitute its separation medium;
placing a significant proportion of the channel at the temperature T2, either prior to or following the introduction of a sample;
introducing a quantity of sample at the inlet of the separating channel;
carrying out the separation at a temperature of the order of T2 in the thermostated portion of the channel; and
detecting the migration of the analytes initially contained in the sample.
This detection involves conventional techniques which fall within the competence of persons skilled in the art and will not therefore be detailed in the present description.
It should be noted that the use of the claimed medium also covers variants in which the temperature is modified during the separation stage since the said variation in temperature comprises a temperature or a range of temperatures T2 at which the thermothickening of the medium is carried out as defined above, as well as the variants in which the above cycle is repeated any number of times, preferably in an automated fashion.
In fact, the invention is particularly advantageous in the case of automated electrokinetic separations since it allows automated filling of the separating channel more easily and more rapidly. Moreover, the introduction of the sample may, in the context of the invention, be carried out before, during or after heating a significant portion of the separating channel to the temperature T2.
The subject of the present invention is also the capillary electrophoresis devices, including those based on chips, using, as separation medium, a medium in accordance with the invention. It is particularly useful in the case of electrophoresis devices termed xe2x80x9cchip-basedxe2x80x9d or etched microchannel-based, since, in general, it is more difficult for these devices to tolerate the application of high pressure values for introducing the separation medium than cylindrical capillaries.
The media according to the invention and the separation methods using these media are particularly advantageous for diagnostic, genotyping, high-throughput-screening and quality control applications, or for detecting the presence of genetically modified organisms in a product.
The figures and examples given below are presented by way of nonlimiting illustration of the present invention.